This project aims to discover small-molecule probes of both mTOR-dependent nutrient-response signaling and histone-mark-dependent chromatin signaling. These probes are expected to illuminate the principles that underlie the nutrient-response and chromatin-signaling cellular networks, and to provide starting points for the discovery of new medicines that act on these networks. These aims will be pursued by the use of (1) global metabolic measurements of cells treated with an mTOR inhibitor, (2) chromatin-state-selective antibodies identified by the use of a new microfluidic/microarray-based technique for screening antibodies, (3) novel high-content screens and gene expression/high-throughput screens for the identification of small- molecule modulators of chromatin-modifying enzymes, and (4) both a novel synthetic chemistry method for targeting small molecules towards chromatin-modifying enzymes and follow-up synthetic chemistry to optimize the properties of the small-molecule probes. Lay language: Recent clinical trials, some successfully completed and others still ongoing, suggest that small molecules that target either of two only recently recognized cellular pathways (one named "nutrient- response" and the other "chromatin") have remarkable potential as life-saving medicines. The recognition of these pathways was made possible by previous research sponsored by this grant. The proposed continuation research aims to discover new small molecules that target new elements of these pathways and to perform experiments that will illuminate their most promising medical applications. PUBLIC HEALTH RELEVANCE The proposed research aims to address the gap between understanding and affecting disease by innovating in chemistry and exploring complex biology with small molecules. The research aims to discover small molecules that modulate two biological processes - stress signaling and chromatin signaling - with considerable promise for medicine. Such small molecules will be used to probe the consequences of modulating key mediators of the processes in order to gain a better understanding of their medical potential as targets for novel therapeutics.